The need for multiplexing techniques in biology is often driven by the fact that test samples are precious and those analyzing them either do not know in advance precisely what to look for or must extract the most information from any single sample. Hence, it is desirable for clinicians and researcher to subject each sample to a large set of probes.
Optical readout is common in biology and can be very effective. However, it is typically limited to a relatively small number of available fluorophores or chromophores (which are referred to collectively as colors). In practice, multiplexing by fluorescence is often limited to 4 or 5 colors, which by traditional methods implies that at most 4 or 5 probes can be detected in a single sample.
The common approach to improving multiplexing in optical methods is to increase the number of available colors. To this end, quantum dots have been developed to provide a larger range of colors. However, in reality, it is difficult to use more than 6 quantum dot colors simultaneously. Another approach is to use mixtures or ratio of fluorophores as new colors. Such methods have extended multiplexing to hundreds of analytes, but due to the size of the labels (e.g., microbeads), the technology has thus far been limited to flow-cytometry based analyses. Yet another approach involves nanostrings, which are essentially short strings of strung-up fluorophores creating visible colorful barcodes. Unfortunately, nanostring readout requires very high-resolution imaging and a special flow apparatus. Further, the nanostrings can only be used in a sample where the probes' targets are sparse, or the barcodes will overlap and create a blur.
A simple workaround for the limited number of colors (e.g., 4 or 5 colors) in optical readouts is to repeat the probing of the same sample with multiple small sets of different probes. For example, the assay can involve probing the sample with 4 different antibodies at a time and imaging after every assay. If the test requires probing the sample with a total of 64 antibodies, the 4-probe procedure would have to be repeated 16 times using the sample. As such, the order of detecting different target analytes in a single sample may need to be prioritized, because some target analytes in the sample can degrade during successive probings. Accordingly, there is still a strong need for accurate and sensitive methods with a high throughput for detection, identification, and/or quantification of target molecules in a sample, e.g., complex mixtures.